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. 2022 Jan 24:2:800898.
doi: 10.3389/ffunb.2021.800898. eCollection 2021.

Development of a Sensitive and Specific Novel qPCR Assay for Simultaneous Detection and Differentiation of Mucormycosis and Aspergillosis by Melting Curve Analysis

Mragnayani Pandey  1 , Immaculata Xess  1 , Janya Sachdev  1 , Usha Yadav  1 , Gagandeep Singh  1 , Dibyabhaba Pradhan  2 , Ashit Bhushan Xess  1 , Bhaskar Rana  1 , Lalit Dar  1 , Sameer Bakhshi  3 , Rachna Seth  4 , Manoranjan Mahapatra  5 , Viveka P Jyotsna  6 , Arun Kumar Jain  7 , Rakesh Kumar  8 , Reshu Agarwal  1 , Prashant Mani  1
Affiliations

Development of a Sensitive and Specific Novel qPCR Assay for Simultaneous Detection and Differentiation of Mucormycosis and Aspergillosis by Melting Curve Analysis

Mragnayani Pandey et al. Front Fungal Biol. .

Abstract

Molecular diagnostic assays can expedite the diagnosis of fungal infections, and subsequently help in early interventions and appropriate management of patients. The aim of this study was to develop a single set of primers for a real-time quantitative polymerase chain reaction (qPCR) assay to detect and identify commonly reported, clinically relevant molds i.e., Aspergillus spp, Mucorales and Fusarium spp., up to genus level by melting curve analysis. This assay was evaluated in whole blood from patients with suspected invasive aspergillosis (IA), and in tissue biopsy, bronchoalveolar lavage (BAL) fluid and other site-specific samples from patients with suspected invasive mucormycosis (IM). The limit of detection (LoD) was determined as 10 copies/μl for all three molds. The mean coefficient of variation (CV) across all sets of intra- and inter-assay data was 0.63% (ranging from 0.42 to 1.56%), showing high reproducibility of the assay. Sensitivity and specificity of the assay were 93.3 and 97.1% respectively for diagnosis of IA, and 99.29 and 83.84% respectively for diagnosis of IM. Fusarium was not detected in any of the clinical samples included and the few laboratory confirmed cases of fusariosis did not meet the inclusion criteria of the study. Hence no ROC curve or cutoff value could be generated for the same. This newly developed qPCR assay therefore appears to be a promising tool in detection of IA and IM.

Keywords: invasive aspergillosis; invasive fusariosis; invasive mucormycosis; melting curve analysis; real-time PCR.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagram of rDNA showing Forward (MF) and Reverse primer (MR) annealing positions. The arrows show the annealing position for primers in the 18S ribosomal subunit.
Figure 2
Figure 2
Variation in melt curve profile of different molds.
Figure 3
Figure 3
Scatter plot of CT value vis Log plasmid copy number. (A) Aspergillus spp (A.flavus). (B) Mucorales. (R.arrhizus) (C) Fusarium spp. (F.solani).
Figure 4
Figure 4
(A) Receiver operator curve plotted significant DNA copy number cut-off for IA cases, classified according to two consecutive GM positivity and radiology findings (B) ROC curve of real time PCR DNA copy number considering direct microscopy as a reference for IM.
Figure 5
Figure 5
Detailed description of the results of conventional and molecular techniques (IM cases).
See this image and copyright information in PMC

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